Quercetin inhibits the epithelial-mesenchymal transition and reverses CDK4/6 inhibitor resistance in breast cancer by regulating circHIAT1/miR-19a-3p/CADM2 axis

Breast cancer (BC) cells have a high risk of metastasis due to epithelial-mesenchymal transition (EMT). Palbociclib (CDK4/6 inhibitor) is an approved drug for BC treatment. However, the drug resistance and metastasis can impair the treatment outcome of Palbociclib. Understanding the mechanisms of EMT and Palbociclib drug resistance in BC is conducive to the formulation of novel therapeutic strategy. Here, we investigated the role of circHIAT1/miR-19a-3p/CADM2 axis in modulating EMT and Palbociclib resistance in BC. circHIAT1 and CADM2 were down-regulated in BC tissues and cell lines, and miR-19a-3p showed an up-regulation. circHIAT1 could interact with miR-19a-3p and suppress its activity, while miR-19a-3p functioned to negatively regulate CADM2. Forced over-expression of circHIAT1 could impaired the EMT status and migratory ability of BC cells, and this effect was inhibited by miR-19a-3p mimic. In addition, we also generated Palbociclib resistant BC cells, and showed that circHIAT1 and CADM2 were down-regulated in the resistant BC cells while miR-19a-3p showed an up-regulation. Forced circHIAT1 over-expression re-sensitized BC cells to Palbociclib treatment. Quercetin, a bioactive flavonoid, could suppressed the migration and invasion of BC cells, and re-sensitized BC cells to Palbociclib. The anti-cancer effect of quercetin could be attributed to its regulatory effect on circHIAT1/miR-19a-3p/CADM2 axis. In vivo tumorigenesis experiment further revealed that quercetin administration enhanced the anti-cancer effect of Palbociclib, an effect was dependent on the up-regulation of circHIAT1 by quercetin. In summary, this study identified quercetin as a potential anti-cancer compound to reverse Palbociclib resistance and impair EMT in BC cells by targeting circHIAT1/miR-19a-3p/CADM2 axis.


Introduction
As the most prevalent malignancy and the top risk factor of cancer-related death in women, breast cancer (BC) is becoming a major health threat to female population [1,2].In spite of Palbociclib drug resistance in BC and suggest quercetin as a potential bioactive compound to reverse Palbociclib resistance in BC treatment.

Clinical sample collection
This study collected breast cancer tissues and para-cancerous specimens from breast cancer patients (N = 20 in each category) in Yunnan University Affiliated Hospital from June 2021 to April 2022.The acquisition of all clinical materials had been approved by the Ethics Committee of Yunnan University Affiliated Hospital (Approval Number: 2020092).In addition, the written form of informed consent were obtained from all the recruited subjects.All the sample handling and data processing steps were following the Declaration of Helsinki.The samples were kept in liquid nitrogen and then at -80˚C for later experiments.

Cell culture
The normal human breast epithelial cell line (MCF-10A) and human BC cell lines (MDA-MB-231 and MCF-7) were procured from Hongshun Biotechnology (Shanghai, China), authenticated using STR profiling, and tested to be mycoplasma-free by the supplier.DMEM medium was utilized to culture the cells, with streptomycin (100 μg/ml), penicillin (100 U/ml), and 10% FBS (Yeasen, Beijing, China).The cells were maintained at 37˚C with 5% CO 2 in a humidified chamber.

Resistant cell line generation
BC cell lines (MDA-MB-231 and MCF-7) were treated with increasing doses of palbociclib (0.1, 0.2, 0.4, 0.8, 1 μM, MedChemExpress, Shanghai, China), with each concentration for 2 to 3 weeks.The drug-containing medium was replenished every three days.Cells were sub-cultured every week when the cell density reached about 80%.After six months, drug-resistant cells were then maintained in the complete medium with 0.8 μM palbociclib.Quercetin was purchased from MedChemExpress (Shanghai, China).

Cell transfection
Lipofectamine™2000 (Invitrogen, Shanghai, China) transfection reagents were used to transfect expression plasmid, siRNA, miRNA mimic or inhibitor into BC cells.Control siRNA and siRNA targeting circHIAT1 were purchased from Genepharma (Shanghai, China).miRNA mimic, inhibitor or the corresponding controls were synthesized by RiboBio Biotech (Guangzhou, China).The empty vector (pcDNA3.1)and circHIAT1 expression vector (pcDNA3.1-circHIAT1)were produced by Sangon Biotech (Shanghai, China).Briefly, cells were seeded in 6-well plates at a density of 5x10^5 cells/well.24 h later, 100 nm of siRNA, miRNA mimic or 6 μg of plasmid was added into 100 μl Opti-MEM 1 I Reduced-Serum Medium (Invitrogen, Shanghai, China), and then 6 μL Lipofectamine 2000 reagent was added in to the medium for 10 min incubation at ambient temperature.The mixture was added to cell culture for 48 transfection before further experimental analysis.

qRT-PCR
Following the manufacturer's instructions, total RNA was extracted using TRIzol (Invitrogen, CA, USA) for quantitative real-time polymerase chain reaction (qRT-PCR).The supernatant was mixed with an equal volume of isopropyl alcohol for 10 minutes before being centrifuged at 12000g/min at 4˚C for 10 minutes.After that the upper layer of clear solution was discarded, the precipitation was washed with 75% ethanol (7500g/min, 4˚C, 5min).The remaining RNA samples were dissolved using DEPC water and the concentration was measured using a spectrophotometer.One Step PrimeScript miRNA cDNA Synthesis Kit (Roche, Laussane, Switzerland) was used to for cDNA synthesis from 1 μg of total RNA.AceQ 1 qPCR SYBR 1 Green Master Mix (Vazyme, Beijing, China) was used for qPCR detection on the 7500 Real Time PCR System (Applied Biosystems, CA, USA).Actb or U6 gene was used as the internal reference for target gene normalization by 2 -ΔΔCt method.All primers were synthesized by Anhui Universal Biosynthesis (Anhui, China).

Western blot
Using a protein extraction kit (Yeasen, Beijing, China), total protein was extracted fro cultured cells and the protein concentration was determined using a BCA assay kit (Beyotime, Beijing, China).10 μg of protein was analyzed using 12% polyacrylamide gels, and then deposited to polyvinylidene fluoride (PVDF) membranes (Sangon Biotech, Shanghai, China).PVDF membranes was blocked with 5% nonfat milk at room temperature for 1 h.The PVDF membrane was then treated overnight at 4˚C with the primary antibodies against the target proteins, followed by a 1-hour incubation with the detection secondary antibody.A Beyo ECL Star kit was employed to detect the protein bands (Beyotime, Beijing, China).The following antibodies were used in this study: N-cadherin, E-cadherin, CADM2, Actin, GAPDH (at 1:1000 dilutions, Cell Signaling Technology, CA, USA).Anti-rabbit IgG HRP (1:5,000; Abcam, Cambridge, UK) was used as the secondary antibody.Protein bands were imaged using a gel imager system (Bio-Rad, CA, United States).

Immunohistochemistry (IHC)
The collected tumor tissues were prepared into 5 μm sections in paraffin.After deparaffinization and rehydration, antigen retrieval was performed by heating the section in citrate unmasking solution (SignalStain 1 Citrate Unmasking Solution (10X) (#14746), Cell Signaling Technologies, CA, USA) at 95˚C for 10 minutes.Sections were cooled on bench top for 30 min, and the washed in dH2O three times for 5 minutes each.The sections were further incubated in 3% hydrogen peroxide for 10 minutes, and then blocked for 1 hour at room temperature in TBST buffer with 5% normal Goat Serum.The following primary antibodies were used for staining: anti-CADM2 and anti-Ki-67 (1:200, Abcam, Cambridge, UK) for 18 hour at 4˚C.The section was then soaked with 500 μL of HRP anti-rabbit secondary antibody (1: 2000 dilution, Cell Signaling Technologies) for 30 minutes at ambient temperature, followed by the addition of 500 μl SignalStain 1 substrate (#8059, Cell Signaling Technologies) for 5 minutes.After rinsing with dH 2 O two times, the section was counter-stained by hematoxylin for 1 minute, and the images were recorded using a light microscope (CX23, Olympus, Japan).

Biotinylated RNA pull-down analysis
Lipofectamine™2000 Reagent was employed to introduce biotinylated miR-NC probe (50 nM) or miRNA mimic into MCF-7 cells.48 h post transfection, the cell lysate from 5x10 5 MCF-7 cells was collected and subjected to the incubation with 100 uL C-1 streptavidin magnetic beads (65001, Life Technologies, Shanghai, China) for 4 h with rotation at 4˚C.After washing, the associated RNA molecules on the beads was extracted using TRIzol reagent, and the relative enrichment of circHIAT1 and CADM2 mRNA was quantified through qRT-PCR.

Cell counting kit-8 (CCK-8) assay
Cells in different experiment groups were inoculated in 96 culture plate (1500 cells per well) and maintained in the incubator for different duration.10 uL CCK-8 reagent (Yeasen, Beijing, China) was then loaded to each well and 3-hour incubation (37˚C, 5% CO 2 ) after being incubated for indicated duration.The culture medium was then discarded and the resulted formazan crystal was dissolved in 150 uL DMSO for 15 minutes.A microplate detector was utilized to record the data at 450nm (OD).

Scratch assay
Cells were grown in a 12-well plate to reach 90% confluency.A sterile pipette tip was utilized to establish a the middle area of cell monolayer.Floating cells were washed away with medium and the remain cells were cultured for another 18 h.The wound closure situation was observed under a light microscope.

Transwell invasion assay
Matrigel matrix (Corning, CA, USA) was applied in a 12-well Transwell chamber and airdried at 37˚C.500 uL serum-free medium containing 2.5x10 5 cells were added into the upper chamber, with 500 uL complete medium being loaded to the lower well.Cells were incubated for 24 hours, and the invading cells on the membrane were fixed with cold 75% ethanol and then labeled with 0.1% crystal violet (Yeasen, Beijing, China) for 30 minutes.At the end of staining, 33% acetic acid was used for decolorization and the images were recorded under a microscope.

Lactate dehydrogenase (LDH) release assay
LDH activity was detected with Lactate Dehydrogenase Assay kit (Beyotime, Beijing, China).Briefly, LDH assay buffer and LDH substrate reaction were mixed and added into each standard of LDH, supernatant from each sample and positive control well.Standard wells = 50 μL standard dilutions; Sample wells = 50 μL samples (adjust volume to 50 μL/well with LDH Assay Buffer); Positive control = 5 μL Positive control and adjust volume to 50 μL/well with LDH Assay Buffer.50 μL of Reaction Mix was added into each standard, sample and positive control sample well.After gentle mix, the output was measured immediately at OD 450 nm using a microplate reader at 37˚C.

Xenograft mouse model
The Department of Experimental Animal model at Yunnan University provided pathogenfree environments for raising BALB/C nude female mice (5 to 6-week-old).The nude mice weighing 30-40 g were housed in pathogen-free conditions on a 12-h light/dark cycle with free access to food and water.The mice were randomly assigned to 5 groups (5 mice in each group): control MCF-7 injection; resistant MCF-7 injection; resistant MCF-7 injection+ quercetin (50 mg/kg); resistant MCF-7 injection+quercetin and control siRNA; resistant MCF-7 injection+quercetin+circHIAT1 siRNA.2×10 6 MCF-7 cells in 0.15 ml PBS was administered subcutaneously at the right flank of each mouse.0.5 mg 17β-estradiol (E2) pellet was implanted into each mouse to support the tumor formation of MCF-7 cells.The administration of quercetin (50 mg/kg/week) and siRNA (500 μg/kg/week) was through intraperitoneal injection.On day 28 following cell injection, all the mice were euthanized by CO 2 asphyxiation and the death was assured by subsequent cervical dislocation.The tumors of terminally dead mice were resected for weight measurement and further analysis.The experimental protocols had been approved by Yunnan University Animal Center Animal Welfare Committee (Approval Number: YNU20200296).

Statistical analysis
Data analyses were performed in SPSS (v23.0) and GraphPad Prism (v6.0).The findings were reported using mean±SD from at least 3 independent experiments.The statistical difference between two groups was compared using unpaired student's t tests.Comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for pairwise comparison.Comparisons of data at multiple time points were examined using two-way ANOVA.P < 0.05 was considered to be statistically different.

circHIAT1 negatively targets miR-19a-3p in BC
We first examined the relative expression of circHIAT1 and miR-19a-3p in tumor specimens and the para-cancerous tissues collected from breast cancer patients in our study (N = 20 in each category).qRT-PCR analysis showed that circHIAT1 was down-regulated, while miR-19a-3p showed an up-regulation in BC tumor samples (Fig 1A and 1B).Through Starbase prediction, there were potential interaction sites between circHIAT1 and miR-19a-3p (Fig 1C).We then performed dual luciferase assay using the reporter vector wild type (WT) or mutated binding sites (MUT).The over-expression of miR-19a-3p using miR-19a-3p mimic could suppress the activity of WT reporter, while there was no inhibition for the MUT reporter (Fig 1D ), indicating the interaction between circHIAT1 and miR-19a-3p at predicted binding sequences.To further demonstrate their physical interaction, we performed RNA pull-down assay using biotin-labeled miR-NC (negative control) or miR-19a-3p probe.We showed that compared to miR-NC probe, biotinylated miR-19a-3p precipitated a significantly higher level of circHIAT1 (Fig 1E).Together, these data suggest the physical association between miR-19a-3p and circHIAT1.Compared to normal breast cell line MCF-10A, circHIAT1 expression level was reduced in BC cell lines (MDA-MB-231 and MCF-7), while miR-19a-3p was up-regulated in BC cell lines (Fig 1F and 1G).We next transfected empty vector or circHIAT1 expression vector in BC cell lines, and the circHIAT1 vector could significantly increase cir-cHIAT1 level (Fig 1H).Upon circHIAT1 over-expression, the level of miR-19a-3p was significantly repressed in BC cell lines, while the expression level of an unrelated miRNA (miR-134-5p, not predicted to interact with circHIAT1) was not affected (Fig 1I).Collectively, these findings indicate that circHIAT1 interacts with miR-19a-3p to negatively regulate its level in BC cells.

circHIAT1/miR-19a-3p/CADM2 axis modulate EMT status and the mobility of BC cells
Entranced mitigation and invasiveness contributed to metastasis in BC cells due to the EMT [5,6].We examined the EMT markers (E-cadherin and N-cadherin) in BC tumor tissues and the para-cancerous samples.The results revealed an increased expression of N-cadherin and a reduced level of E-cadherin in the tumor samples, indicating the enhanced mesenchymal feature in BC.Meanwhile, CADM2 expression also displayed a down-regulation in the tumor samples (Fig 3A).We next examined whether circHIAT1/miR-19a-3p axis impacts on the migratory and invasion ability through EMT in BC cells.circHIAT1 over-expression reduced N-cadherin level and promoted the expression of E-cadherin, while the co-administration of miR-19a-3p mimic abrogated the effects of circHIAT1 over-expression; however, the co-transfection of CADM2 vector reversed the effect of miR-19a-3p mimic (Fig 3B).CCK-8 assay revealed that circHIAT1 over-expression caused the retardation of cell proliferation, which could be rescued by miR-19a-3p mimic; the co-transfection of CADM2 vector repressed the proliferation (Fig 3C).Consistent with the change of EMT markers, cir-cHIAT1 over-expression suppressed cell migration and invasion in the cell scratch and transwell invasion assays.The co-transfection of miR-19a-3p mimic largely rescued the migration and invasion abilities, while the effect of miR-19a-3p mimic was abolished by CADM2 expression (Fig 3D and 3E) These data imply that circHIAT1/miR-19a-3p axis modulates cell migration and invasion by regulating EMT in BC cells through targeting CADM2.

Quercetin suppresses migration and invasion of BC cells at sub-toxic dose
Quercetin was reported to have anti-cancer effect in BC cells [20,21], while its potential effect on EMT has not been investigated.We first examined the cytotoxicity of quercetin on MDA-MB-231 and MCF-7 cell line.Cells were treated with 0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM quercetin for 48 hours and the cell toxicity was assessed by LDH assay.The results showed that quercetin showed significant cytotoxicity above 20 μM, while at 10 μM it did not exert significant toxicity on BC cells (Fig 4A).We therefore selected 10 μM as the sub-toxic concentration in following experiments, which excluded the potential toxic effects of quercetin.At 10 μM, CCK-8 assay demonstrated that quercetin could show antiproliferation effect on BC cells (Fig 4B).Besides, quercetin also suppressed the migration and invasion in BC cells at the sub-toxic concentration (Fig 4C and 4D).Western blot analysis of E-cadherin and N-cadherin also showed that quercetin repressed EMT process in BC cells (Fig 4E).We also analyzed the impact of quercetin on the expression of cir-cHIAT1, miR-19a-3p and CADM2.Intriguingly, quercetin treatment promoted the expression of circHIAT1 and CADM2, but reduced miR-19a-3p level in BC cells (Fig 4E and 4F).These results prompted us to further study whether circHIAT1/miR-19a-3p axis mediates the effect of quercetin.

The anti-cancer effect of quercetin depends on circHIAT1/miR-19a-3p axis
In MCF-7 cells, we treated the samples with 10 μM quercetin in the presence of control siRNA, circHIAT1 siRNA or circHIAT1 siRNA plus miR-19a-3p inhibitor.qRT-PCR analysis showed that quercetin promoted circHIAT1 expression and circHIAT1 siRNA repressed its level.Quercetin reduced miR-19a-3p expression, and the transfection of circHIAT1 siRNA increased miR-19a-3p level; and miR-19a-3p inhibitor could also reduced miR-19a-3p level upon circHIAT1 silencing (Fig 5A).As expected, quercetin repressed the expression of N-cadherin and promoted E-cadherin level, which could be impaired by circHIAT1 siRNA; the cotransfection of miR-19a-3p inhibitor abrogated the effect of circHIAT1 siRNA (Fig 5B).Silencing circHIAT1 also abolished the inhibitory effect of quercetin on cell proliferation, while the effect of circHIAT1 knockdown was attenuated by miR-19a-3p inhibition (Fig 5C).In agreement with the change of EMT markers, silencing circHIAT1 rescued cell migration and invasion upon quercetin treatment, while the effect of circHIAT1 knockdown was abolished by miR-19a-3p inhibition (Fig 5D and 5E).Thus, the anti-cancer effect of quercetin depends on circHIAT1/miR-19a-3p axis.

Quercetin re-sensitizes BC cells to CDK4/6 inhibitor Palbociclib by targeting circHIAT1
To investigate the effect of quercetin on Palbociclib drug resistance, we continuously exposed BC cells to increasing doses of Palbociclib.In contrast to the parental cell lines, the resistant MCF7 and MDA-MB-231 cells were able to proliferate in the presence of different doses of Palbociclib (Fig 6A).In the drug-resistant cells, we also found that circHIAT1 and CADM2 became down-regulated while miR-19a-3p was up-regulated (Fig 6B).We therefore investigated whether circHIAT1 dysregulation also contribute to Palbociclib resistance.Drug-resistant cells were transfected with empty vector or circHIAT1 expression vector.In the presence of 0.8 μM Palbociclib, drug-resistant cells continued to proliferate while circHIAT1 overexpression repressed the proliferation (Fig 6C).In addition, LDH assay revealed that cir-cHIAT1 over-expression re-sensitized the resistant BC cells to Palbociclib induced cytotoxicity (Fig 6D).Therefore, circHIAT1 down-regulation contributes to Palbociclib drug resistance.To further study whether quercetin impacts on Palbociclib resistance, drug-resistant cells were treated with 10 μM quercetin for 24 hours before Palbociclib treatment.Co-treatment with quercetin abrogated the cell proliferation ability of BC cells under 0.8 μM Palbociclib, and also induced cell cytotoxicity (Fig 6E and 6F).Since quercetin treatment increased circHIAT1 level in BC cells (Fig 4F ), we silenced circHIAT1 to examine whether circHIAT1 up-regulation contributes to the effect of quercetin.circHIAT1 knockdown by siRNA suppressed the effect of quercetin on sensitizing BC cells to Palbociclib treatment (Fig 6G and 6H).Together, these data suggest that quercetin is able to re-sensitize drug-resistant BC cells to CDK4/6 inhibitor Palbociclib by targeting circHIAT1.

Quercetin promotes Palbociclib sensitivity of drug-resistant BC cells in mouse xenograft model
In order to evaluate the in vivo effect of quercetin treatment on drug-resistant BC cells, nude mice were injected with drug-sensitive or drug-resistant MCF-7 cells.The drug-resistant group was also administered with quercetin, or quercetin and circHIAT1 siRNA.All the mice were treated with Palbociclib to assess the drug sensitivity.As expected, drug-resistant MCF-7 cell injection led to large tumor size and heavier tumor weight when compared to the mice injected with drug-sensitive cells; quercetin administration could retard tumor growth of drug-resistant MCF-7 cells, and this effect was attenuated by circHIAT1 siRNA (Fig 7A and  7B).The results of IHC staining in the tumor sections showed that the cells expressing the proliferation marker Ki67 were increased in drug resistant-tumor section, and the number of Ki67-positive cells was decreased after quercetin treatment, but the knockdown of circHIAT1 could reverse the effect of quercetin (Fig 7C).Further, qRT-PCR analysis revealed that cir-cHIAT1 and CADM2 were down-regulated in drug-resistant cells, and quercetin treatment increased their expression.These effects were inhibited by circHIAT1 knockdown, while miR-19a-3p showed an opposite expression pattern (Fig 7D).Furthermore, the analysis of EMT markers demonstrated that, the elevated expression of mesenchymal marker N-cadherin in drug-resistant tumor was suppressed by quercetin treatment, and the effect of quercetin was partially abrogated upon circHIAT1 silencing.The expression pattern of E-cadherin exhibited an opposite trend to that of N-cadherin (Fig 7E).Thus, quercetin also suppresses the mesenchymal state of BC cells in vivo through modulating circHIAT1.Collectively, these results imply that quercetin boosts the anti-cancer effect of Palbociclib on drug-resistant BC cells by targeting circHIAT1.

Discussion
As a major cause of cancer-related death and a common type of cancer in women, BC appears in a wide age range from 20-60 years old [26].The prevalence of BC has increased noticeably over the past few years [27].Although the advanced diagnosis and treatment solutions improved the overall survival of BC patients [28,29], a large portion of patients suffers from dismal prognosis due to distance metastasis, drug resistance and cancer recurrence [30,31].In this study, we demonstrated the roles of circHIAT1/miR-19a-3p/CADM2 axis in modulating the EMT and drug resistance of Palbociclib in BC cells, and showed that quercetin can act as a potential bioactive compound to reverse Palbociclib resistance in BC treatment.
There is a strong relationship between therapeutic resistance and EMT induction in cancer [32].CDK4/6 inhibitors have been developed as therapeutic agents for treating a wide range of malignancies [33].Clinical evidence showed that about only 10% of the patients show primary resistance to CDK4/6 inhibitors, and the number of patients with failed treatment increase with prolonged drug administration [34].Palbociclib has been implemented as a first-line therapy along with other endocrine drugs for BC treatment [35].CDK4/6 inhibitor resistance is related to EMT through activating the PI3K/AKT/mTOR and TGF-β-Smad signaling pathways [11].We showed that circHIAT1 and CADM2 becomes down-regulated while miR-19a-3p is over-expressed in Palbociclib resistant BC cells.circHIAT1 over-expression could impair EMT of BC cells and re-sensitizes drug-resistant BC cells to Palbociclib treatment in vitro and in vivo.We also demonstrated the molecular interplay of circHIAT1, miR-19a-3p and CADM2 in BC cells.Theretofore, circHIAT1/miR-19a-3p/CADM2 may play a critical role in dictating EMT process and Palbociclib drug resistance in BC cells.
Cell Adhesion Molecule 2 (CADM2) is engaged in homo-and heterophilic interactions with the other nectin-like family members.CADM2 over-expression could suppressed the EMT and cell proliferation of esophageal squamous cell carcinoma cells by repressing AKT signaling pathway [36].CADM2 dysregulation has been also implicated in the regulation of EMT process of other types of cancers [37,38].In this study, we reported that down-regulation of CADM2 is associated with entranced EMT process in Palbociclib-resistant BC cells.We also provide evidence that circHIAT1/miR-19a-3p axis regulates CADM2 in BC cells.The mechanisms by which CADM2 regulates Palbociclib resistance in BC cells need to be further clarified in future work.
Quercetin has been reported to induced apoptosis and necrosis in BC cells [20,21,39].However, in this study, we demonstrated that at sub-toxic dose, quercetin treatment could impair EMT and re-sensitize drug-resistant BC cells to Palbociclib treatment.This effect seems to be dependent on its effect on inducing circHIAT1 up-regulation, which then impinges on miR-19a-3p and CADM2 expression.Many studies have reported the role of miR-19a-3p role in BC.For instance, Xiang et al reported that miR-19a-3p up-regulation promotes the migration and EMT in BC cells, and dampens the sensitivity to Letrozole [23].There is also evidence that miR-19 enhances cell invasion, proliferation, migration, and EMT in osteosarcoma and lung cancer cells [40,41].Jiang et al. highlighted the role of miR-19a-3p in promoting chemoresistance and metastasis in hepatocellular carcinoma by targeting PTEN/AKT pathway [42].Therefore, our data are consistent with the notion that miR-19a-3p serves as an oncogenic factor to promote the progression of BC cells by augmenting EMT and drug resistance.However, how quercetin induces circHIAT1 up-regulation in BC cells requires future investigation.
There are very few reports on the relationship between circHTAT1 and miR-19a-3p.According to Hu et al., miR-19a-3p was extensively expressed in cervical and circHIAT1 was a sponge factor of miR-19a-3p to suppress its activity [43].The tumor-suppressive function of circHIAT1 was also reported in hepatocellular carcinoma [44].Yang et al. showed that miR-19a-3p was up-regulated significantly in renal carcinoma and CADM2 was a direct target of miR-19a-3p [24].The knockdown of miR-19a-3p suppressed the invasion, proliferation, and EMT of renal carcinoma cells.In current study, we found that quercetin suppressed the migration, invasion, and EMT in BC cells, while circHIAT1 knock-down impaired the effect of quercetin.As far as we know, this is the first report regarding the role of quercetin on the EMT and CDK4/6 inhibitor drug resistance in BC.Although palbociclib was known to inhibit the EMT and metastasis in BC [9], the prevalent drug resistance undermines its treatment outcome.Our findings suggest that the joint application of quercetin and palbociclib might have synergistic effect on suppressing the EMT and metastasis in BC.

Conclusions
Taken together, our study identified quercetin as a potential anti-cancer compound to reverse Palbociclib resistance and impair EMT in BC cells by targeting circHIAT1/miR-19a-3p/ CADM2 axis.These data not only provide novel insights into the regulation of EMT and invasive features of BC cells, but also hint on the potential of joint application of quercetin and palbociclib to overcome drug resistance.Future work needs to clarify the mechanisms by which quercetin impinges on circHIAT1/miR-19a-3p/CADM2 axis.
TargetScan tool was used to predict the potential targets of miR-19a-3p.The results indicated that there were potential binding sites for miR-19a-3p at the 3'UTR of CADM2 mRNA (Fig2A).Dual luciferase reporter assay confirmed that miR-19a-3p interacts the 3'UTR of CADM2 mRNA at predicted wildtype sequences (Fig2B).RNA pull-down analysis further revealed the enrichment of CADM2 mRNA by biotin-labeled miR-19a-3p probe (Fig2C).Besides,

Fig 7 .
Fig 7. Quercetin boosts the anti-cancer effect of Palbociclib on BC cells in mouse xenograft model.To evaluate the in vivo effect of quercetin on drug-resistant BC cells, nude mice were injected with drug-sensitive or drug-resistant MCF-7 cells.The drug-resistant group was also administered with quercetin, or quercetin and circHIAT1 siRNA.All the mice were treated with Palbociclib to assess the drug sensitivity.(A).Images of xenograft tumors in each group; (B).Summary of tumor weight; (C).IHC staining of Ki-67 cell proliferation marker in tumor sections; (D).qRT-PCR was performed to analyze circHIAT1, miR-19a-3p and CADM2 expression in the tumor samples of each group.(E).Western blot analysis of N-cadherin and E-cadherin protein levels in the tumor samples of each group.*p<0.05;**p<0.01;***p<0.001;****p<0.0001.https://doi.org/10.1371/journal.pone.0305612.g007